1. Technical Field
The present invention relates to a method of amplifying a nucleic acid sequence that is useful in the field of genetic engineering. More particularly, the present invention relates to a method of amplifying a nucleic acid sequence that utilizes a strand displacement reaction, and a mutation detection method using the same.
2. Background Art
In the field of genetic engineering, an assay based on the complementation of nucleic acid sequences is known as a method that allows genetic features to be analyzed directly. In such an assay, when a target (aimed) gene is present only in a small amount in a sample, the detection thereof generally is not easy. It therefore is necessary to amplify the target gene itself beforehand.
The amplification of the target gene (nucleic acid amplification) mainly is carried out by an enzymatic method with the use of DNA polymerase. Major examples of such an enzymatic method include the polymerase chain reaction method (PCR method; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159) and the reverse transcription PCR method (RT-PCR method; Trends in Biotechnology 10, pp 146-152, 1992) that is a combination of the PCR method and a reverse transcriptase reaction. These methods allow a target gene derived from DNA or RNA to be amplified by repeating a reaction that consists of three steps. The three steps include: the dissociation (denaturation) of a double-stranded nucleic acid into a single-stranded nucleic acid to serve as a template; the annealing of a primer to the single-stranded nucleic acid; and the synthesis (extension) of a complementary strand from the primer. These methods require the repetition of three steps in total in which the reaction solution is adjusted to a temperature suitable for each reaction in the three steps described above.
Furthermore, EP 0320308 A discloses the ligase chain reaction method (LCR method). In this method, a known gene sequence is amplified by conducting a two-step temperature cycling reaction (a heating-cooling repeated reaction) using a thermostable DNA ligase.
However, the methods described above require the use of an expensive thermal cycler that can control temperature strictly over time in a wide range of temperatures. Since the above-mentioned reactions are conducted under two to three temperature conditions, time is required to make an adjustment to each reaction temperature. Hence, the time required for the adjustment increases as the number of cycles increases.
In order to solve the above-mentioned problems, nucleic acid amplification methods have been developed that can be carried out isothermally. Examples of such methods include: the strand displacement amplification (SDA) method described in JP7(1995)-114718B; the self-sustained sequence replication (3SR) method; the nucleic acid sequence based amplification (NASBA) method described in Japanese Patent No. 2650159; the transcription-mediated amplification (TMA) method; the Q-beta replicase method described in Japanese Patent No. 2710159; various improved SDA methods described in U.S. Pat. No. 5,824,517, W099/09211, and WO 95/25180; the loop-mediated isothermal amplification (LAMP) method described in WO00/28082; and the isothermal and chimeric primer initiated amplification of nucleic acids (ICAN method) described in WO02/16639. The reactions in all the steps involved in the isothermal nucleic acid amplification methods described above proceed simultaneously in a reaction mixture maintained at a constant temperature.
In the SDA method, a target nucleic acid (and a complementary strand thereto) in a sample can be amplified through the displacement of a double strand mediated by a DNA polymerase and a restriction endonuclease in a system where DNA is amplified finally. This method requires four primers, two of which need to be designed so as to contain a recognition site for the restriction endonuclease. Furthermore, this method requires a modified deoxynucleotide triphosphate as a substrate for nucleic acid synthesis. The modified deoxynucleotide triphosphate is, for instance, a deoxynucleotide triphosphate in which the oxygen atom of the phosphate group located at the alpha position of the triphosphate moiety has been substituted by a sulfur atom (S). Accordingly, this method requires high running cost. Moreover, in this method, the amplified nucleic acid fragment contains a modified nucleotide such as an alpha-S-displaced deoxynucleotide. Hence, for example, when the amplified fragment is subjected to a restriction enzyme fragment length polymorphism (RFLP) assay, the amplified fragment cannot be cleaved by a restriction enzyme and thus such an assay cannot be conducted in some cases.
The improved SDA method described in U.S. Pat. No. 5,824,517 requires a chimeric primer that is composed of RNA and DNA, with the DNA being located on the 3′ end side. Such a chimeric primer that is composed of RNA and DNA is difficult to synthesize. Furthermore, in order to handle a primer containing RNA, professional skills are required. Furthermore, the improved SDA method described in W099/09211 requires a restriction enzyme that produces a 5′ protruding end. Moreover, the improved SDA method described in WO95/25180 requires at least two pairs of primers. Hence, these methods require high running cost.
In the ICAN method, it is necessary to use a chimeric primer as well as an RNaseH. The chimeric primer is composed of RNA and DNA, with the RNA being located on the 3′ end side. The RNaseH cleaves the RNA moiety located at the 3′ end of the primer. Accordingly, an increased number of reagents are used and longer processing time also is necessary. Thus the ICAN method is not suitable for processing a large amount of samples.
In the LAMP method, four primers are necessary. They recognize six regions, so that a target gene can be amplified. That is, in this method, a first primer anneals to a template strand to cause extension, and then the extended strand produced by the first primer separates from the template strand due to the strand displacement reaction caused by a second primer designed upstream from the first primer. At this time, a stem-loop structure is formed in the 5′ end portion of the extended strand due to the structure of the first-primer extension product that has been removed. Similar reactions occur in the other strand of the double-stranded nucleic acid or on the 3′ end side of the first-primer extension product that has been removed. These reactions are repeated and thereby the target nucleic acid is amplified. Hence, in the LAMP method, the action mechanism of the amplification reaction is complicated and furthermore, six regions must be selected. This makes it difficult to design the primers. Moreover, two of the four primers are required to be relatively long chain primers. Accordingly, the synthesis and purification of the primers are complicated and reagents are difficult to prepare.
There is a need for a nucleic acid amplification method that can be carried out at lower running cost and that allows a nucleic acid fragment obtained thereby further to be used for genetic engineering treatments. Particularly, an isothermal nucleic acid amplification method is desired that allows amplification to be conducted quickly with a pair of primers.
When a single nucleotide mutation that is present in a target nucleic acid is to be detected using such an amplification method as described above, various problems have arisen. For instance, in the mutation detection by the PCR-SSP method based on the PCR method, a primer is used that has a nucleotide associated with a mutation at the 3′ end, and the mutation is detected depending on the presence or absence of an amplification product. In an amplification reaction caused by such a primer, however, even when the nucleotide associated with a mutation and the nucleotide located at the 3′ end of the primer are not complementary to each other, an extension reaction may occur erroneously. In the PCR method, a double-stranded nucleic acid synthesized through the extension reaction of a primer is used as a new template. In this case, the sequence to which the next new primer anneals is not the nucleotide sequence that has been contained originally in the sample but a copy of the primer sequence. Accordingly, even if the complementary strand synthesis of a wrong region occurs only once, the wrong region is amplified one after another. Hence, amplification products other than those intended to be obtained are produced readily. Thus, it is difficult to detect a single nucleotide mutation correctly.
Furthermore, in the PCR-SSO method, a probe DNA that can hybridize to a region containing a mutated site is brought into contact with a target amplification product obtained through amplification performed by the PCR method. Then it is observed whether hybridization occurs or not and thereby the presence or absence of a mutation in the target amplification product is determined. In this method, however, beside the longer hybridization reaction time, problem is a specificity. For example, nonspecific hybridization may take place depending on the stringency of the reaction solution. Thus it is not easy to check the mutation of a single base accurately.
Another mutation detection UCAN method is based on ICAN method where a DNA-RNA-DNA chimeric primer is used that contains a nucleotide associated with a mutation in a RNA portion. The DNA located at the 3′ end of this chimeric primer has been modified chemically so that no extension reaction occurs therefrom. In the case where an amplification reaction is conducted in a reaction solution containing such a chimeric primer and RNaseH, the RNaseH cleaves the RNA portion only when the sequence of the chimeric primer and that of the template match completely with each other. In that case, an extension reaction starts from the 3′ end of a newly produced primer and thereby the template DNA is amplified. On the other hand, when the sequence of the chimeric primer and that of the template DNA do not match with each other, i.e. when a mutation exists, the RNA portion is not cleaved by RNaseH. In this case, the 3′ end of the chimeric primer remains chemically modified. Thus DNA amplification does not occur. However, in both the ICAN and UCAN methods, the amplification is performed through specific hybridization to two regions of a template as in the conventional PCR method. Hence, there is a problem in the specificity. Accordingly, after the amplification, it is necessary further to check whether the amplification product obtained is a target one. Thus it takes a long time until the examination results are obtained. In addition, the syntheses of the modified primer, the chimeric primer, etc. are complicated.
In the LAMP method, at least four primers are necessary. Accordingly, for example, a primer dimer tends to be produced. Furthermore, since six specific regions are necessary, the primers are very difficult to design. Hence, it takes a long time to study the conditions that improve the specificity of nucleic acid amplification, for example. Moreover, in the mutation detection that is performed by the LAMP method described in WO01/034838, a mutation is recognized at the 3′ end of a dumbbell structure that is an amplification product produced during the amplification. In this method, it is considered that when a mutation exists at the 3′ end of the dumbbell structure, the extension reaction stops occurring therefrom, which inhibits the target region from being amplified. However, like the case of the PCR-SSP method, a mismatch of one base located at the 3′ end does not always stop the extension reaction. Even when no amplification occurs from the 3′ end of the dumbbell structure, the amplification product itself already has formed a dumbbell structure. Accordingly, a stem-loop structure of itself has been formed, and therefore the primer anneals to the loop structure portion. Thus the extension reaction that occurs from the 3′ end of the primer always is conducted. It therefore is very difficult to identify the single nucleotide mutation based on whether or not the amplification has occurred.
Recently, great importance has been attached to diagnostic techniques for quickly detecting gene information such as gene insertion, gene deletion, etc. Particularly, importance is attached to a technique for analyzing a target gene easily, quickly, and accurately, for instance, a technique for specifically detecting a gene marker or mRNA that is expressed specifically to cancer cells, etc.
When mRNA is to be detected that has been expressed specifically to only a specific cell type, such as a cancer cell, a nucleic acid sample that commonly is used includes not only target mRNA but also genomic DNA intermingled therein. The nucleotide sequence of mRNA is one obtained by removing sequences of some intron portions from the nucleotide sequence of genomic DNA. One intron generally has a strand length of several bases to several hundreds of bases. When using such a nucleic acid sample as a template and a primer like the one that is used in the PCR method, both mRNA and genomic DNA can serve as templates. Hence, amplification occurs from both templates. Even when the primer is designed so as to allow mRNA to be amplified specifically, amplification generally occurs not only from mRNA but also from genomic DNA in a nucleic acid sample containing genomic DNA intermingled therein, since the sequence of mRNA is a part of the sequence of genomic DNA. Accordingly, mRNA cannot be amplified specifically. Furthermore, it is considered to be very difficult to amplify such mRNA accurately and quantitate mRNA present in the sample. Moreover, in the case of amplifying a target nucleic acid in which an insertion or deletion of several bases to several hundreds of bases exists and then determining the presence or absence of the amplification product thereof, it is very difficult to recognize a small difference in size when the band of a target amplification product is checked by the electrophoresis method that has been employed conventionally. When gene diagnosis is performed at a clinical site, it is necessary to process many samples simply and efficiently in a short time. Hence, the conventional methods cannot deal with this sufficiently.